PROJECT SUMMARY/ABSTRACT CORE C ? GENOME ENGINEERING CORE Core C is focused on providing the most up-to-date genome engineering technology for studies of cardiac development. We will develop and adopt emerging technology for CRISPR/Cas9-mediated gene deletions, single-base changes, transgene insertions, and epigenetic remodeling. The PPG proposal utilizes advanced genome engineering techniques. We will specifically provide genome engineering services of human iPSCs to efficiently deliver engineered iPSCs for cardiac differentiation. The Genome Engineering Core will also provide epigenome editing methods, that modify the cell's genome without cutting DNA. The CRISPR- associated nuclease (Cas9) has been modified so that the nuclease is inactive in making ?dCas? that can now be used to carry and localize a wide variety of bioactive molecules to any location in the genome. We pioneered the use of CRISPRi that silences gene expression in iPSCs, and are involved in developing better methods that activate gene expression (CRISPRa). Our team is an established leader in genome engineering, and has made efforts to improve every aspect of genome editing in human iPSCs to benefit the PPG investigators in their efforts to unravel the molecular basis of congenital heart disease. In the last 5 years, we made >50 different genetically modified human iPSC lines with point mutations that exactly mimic the disease mutations, generate insertions/deletions (indels) for gene knockouts, or introduce endogenous gene tags for molecular studies of protein function (Miyaoka et al., 2014, Huebsch et al., 2015, Mandegar et al., 2016, Judge et al., 2017). The Genome Engineering Core will adopt the latest methods for genome engineering, such as the use of CRISPR/Cas9 RNP-protein complexes (RNP) to introduce insertion/deletions (indels), or delete discrete portions of genes to inactivate them in iPSCs as well as in iPSC-derived cardiomyocytes, since RNP-mediated genome editing is more efficient and accurate in our experience. In addition, the Genome Engineering Core will provide genome engineering services for the insertion of transgenes at endogenous loci, as well as develop new CRISPR methods for the PPG investigators to further investigate the cardiac interactome. The Genome Engineering Core will develop a pipeline to deliver high-quality engineered iPSCs to the PPG projects with continuously updated techniques, to answer vital questions in heart development.